This unit provides a step-by-step protocol for constructing cell type- and mitochondria-targeted GCaMP genetically encoded Ca2+ indicators (GECIs) for mitochondrial Ca2+ imaging in astrocytes. Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Mitochondrial Ca2+ overload contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Live-cell mitochondrial Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics and thus cell physiology and pathology. We implement astrocyte-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s). By loading X-Rhod-1 into astrocytes, we can simultaneously image mitochondrial and cytosolic Ca2+ signals. This protocol provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria. © 2018 by John Wiley & Sons, Inc.